The long-term objectives are to develop an understanding of the cellular and subcellular events pertinent to the regulation of gene activity in mammalian development, and ultimately to understand the molecular processes that control gene expression in normal and abnormal developmental processes. The approach is to exploit the dichotomy in patterns of X-chromosome inactivation (random vs. determinate) between eutherian and metatherian mammals as comparative probes into patterns of X-linked virginiana (Virginia opossum: the only metatherian known to exhibit variation at more tan one X-linked gene locus) and Monodelphis domestica (gray, short-tailed opossum), will serve as model systems to establish the patterns and timing of X-chromosome replication and the expression of paternally and maternally derived X-linked genes during critical stages of embryogenesis and early postnatal development. Specifically, the aims are (1) to determine the relative levels of expression of the Gpd and Pgk-A genes (encode glucose-6-phosphate dehydrogenase and phosphoglycerate kinase-A, respectively on the maternal and paternal X chromosomes in tissues and organs of newborn pouch young, and in tissues, organs, and extraembryonic membranes of mid to late gestational embryos of D. virginiana; (2) to determine whether differential expression of the maternal and paternal Gpd and Pgk-a genes occurs in early embryonic stages of D. virginiana, and if so, when it can first be detected; and (3) to establish whether a late replicating X chromosome (indicative of the inactive state) is present in embryonic and extraembryonic tissues during early and mid gestational stages in D. virginiana and M. domestica, and if so, to relate replication timing to pattern of Gpd and Pgk-A expression. FEmale embryos of known developmental stage, and in the case of D. virginiana, specific Gpd and Pgk-A genotypes, will be produced by laboratory crosses. Electrophoretic methods will be used to deduce, in D. virginiana, the relative levels of expression of the maternal and paternal Gpd and Pgk-A alleles in 1) whole early embryos, 2) extraembryonic membranes and embryo proper in older embryos, and 3) various tissues and organs of newborn pouch young. the 5-BrdU-labeling technique will be used to establish the presence/absence of a late replicating X chromosome in cells of embryonic stages in both species.